It is already known that Toll genes are necessary for determining the dorsoventral axis in the embryogeny of Dorsophilia (Cell 52, 269-279, 1988, Annu Rev. Cell Dev. Biol. 12, 393-416, 1996) and for antifungal immune responses in the adult fly (Cell 86, 973-983, 1996).
It has been shown that the Toll is a Type I transmembrane receptor comprising leucine-rich repeat (LRR) in extracellular domains, and its intracellular domains are highly homologous to the intracellular domains of mammalian interleukin-1 receptor (IL-1R) (Nature 351, 355-356, 1991, Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996, J. Leukoc. Biol. 63, 650-657, 1998).
Recently, mammalian homologs of Toll called Toll-like Receptor (TLR) have been identified, and six members of the family such as TLR2 and TLR4 have been reported (Nature 388, 394-397, 1997, Proc. Natl. Acad. Sci. USA 95, 588-593, 1998, Blood 91, 4020-4027, 1998, Gene 231, 59-65, 1999). It is known that a member of the TLR family mediates MyD88, an adopter protein as IL-LR is, recruits IL-LR binding kinase (IRAK), activates TRAF6, and activates downstream NF-κ B (J. Exp. Med. 187, 2097-2101, 1998, Mol. Cell 2, 253-258, 1998, Immunity 11, 115-122, 1999). It is also thought that the role of the TLR family in mammals is related to innate immune recognition as a pattern recognition receptor (PRR) recognizing bacterial common components (Cell 91, 295-298, 1997).
It is well known that one of the pathogen-associated molecular patterns (PAMP) recognized by the PRR mentioned above is lipopolysaccharide (LPS), which is a main component of the outer membrane of Gramm-negative bacteria (Cell 91, 295-298, 1997), the LPS stimulates a host cell to produce various inflammatory cytokines such as TNF α, IL-1 or IL-6 in the host cell (Adv. Immunol. 28, 293-450, 1979, Annu. Rev. Immunol. 13, 437-457, 1995), and the LPS captured by LPS-binding protein (LBP) is transferred to CD 14 on the surface of a cell (Science 249, 1431-1433, 1990, Annu. Rev. Immunol. 13, 437-457, 1995). The present inventors generated TLR4 knockout mice and reported that the TLR4 knockout mice lack the ability to respond to LPS, a main component of the outer membrane of the Gram-negative bacteria (J. Immunol. 162, 3749-3752, 1999), and also generated TLR2 knockout mice and reported that macrophages derived from TLR2 knockout mice showed low levels of response to cell wall of Gram-negative bacteria or peptidoglycan, a component of the Gram-negative bacteria (Immunity 11, 443-451, 1999).
On the other hand, from the fact that the oligonucleotides comprising bacterial DNA (DNA derived from bacteria) or an unmethylated CpG sequence stimulate immune cells of mice or human (Trends Microbiol. 4, 73-76, 1996, Trends Microbiol. 6, 496-500, 1998), and stimulate a T helper 1 cells (Th1)-like inflammatory response dominated by the release of IL-12 and IFNγ (EMBO J. 18, 6973-6982, 1999, J. Immunol. 161, 3042-3049, 1998, Proc. Natl. Acad. Sci. USA 96, 9305-9310, 1999), it is advocated that the oligonucleotides comprising CpG sequence are possibly used as an adjuvant in vaccine strategies including vaccines to cancer, allergy and infectious diseases (Adv. Immunol. 73, 329-368, 1999, Curr.Opin. Immunol. 12, 35-43, 2000, Immunity 11, 123-129, 1999). Although its effects have been expected in the clinical practice in this way, the molecular mechanism by which bacterial DNA comprising an unmethylated CpG sequence activates immune cells is unclear.
Although the DNA derived from bacteria comprising an unmethylated CpG motif activates immune cells significantly and induces response by Th1 as mentioned above, the activities at the molecular level are not well understood. The goal of the present invention is to provide a receptor protein TLR9, a member of TLR family specifically recognizing bacterial DNA comprising an unmethylated CpG sequence, the DNA encoding it, and the artificial animal models useful in examining response of host immune cells to bacterial infectious diseases, which elucidate effects of oligonucleotides comprising an unmethylated CpG sequence of bacterial DNA at the molecular level.
As a member of the mammalian TLR family, a pattern recognition receptor recognizing common structures of bacteria, relevant to innate immune recognition, six members (TLR1 to 6) have been publicized until now (Nature 388, 384-397, 1997, Proc. Natl. Acad. Sci. USA, 95, 588-593, 1998, Gene 231, 59-65, 1999), and TLR7 and TLR8, two novel members, are registered in GenBank (Registration No: AF240467 and AF246971). Although full-length cDNA is also found out for TLR9, and is registered in GenBank (Registration No: AF245704), its function has not been known.
The present invention relates to DNA encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence (“1”), the protein according to “1” wherein a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is either of the following proteins (a) or (b): (a) a protein comprising the sequence of amino acids shown in Seq. ID No: 2, or (b) a protein comprising a sequence of amino acids wherein one or more of amino acids are deleted, substituted, or added in the sequence of amino acids shown in Seq. ID No: 2, and having reactivity against bacterial DNA having an unmethylated CpG sequence (“2”), the DNA according to “1” comprising the sequence of bases shown in Seq. ID No: 1 or its complementary sequence, or part or whole of the sequences (“3”), the DNA according to “1” which hybridizes with the DNA comprising a gene according to “3” under a stringent condition (“4”), the protein according to “1” wherein a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is either of the following proteins (a) or (b): (a) a protein comprising the sequence of amino acids shown in Seq. ID No: 4, or (b) a protein comprising a sequence of amino acids wherein one or more of amino acid are deleted, substituted, or added in the sequence of amino acids shown in Seq. ID No: 4, and having reactivity against bacterial DNA having an unmethylated CpG sequence (“5”), the DNA according to “1” comprising the sequence of bases shown in Seq. ID No: 3 or its complementary sequence, or part or whole of the sequences (“6”), and the DNA according to “1” which hybridizes with the DNA comprising the gene according to “6” under a stringent condition (“7”).
The present invention also relates to a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence (“8”), the protein according to “8” comprising the sequence of amino acids shown in Seq. ID No: 2 (“9”), the protein according to “8” comprising a sequence of amino acids wherein one or more of amino acids are deleted, substituted or added in the sequence of amino acids shown in Seq. ID No: 2 (“10”), the protein according to “8” comprising the sequence of amino acids shown in Seq. ID No: 4 (“11”), and the protein according to “8” comprising a sequence of amino acids wherein one or more of amino acids are deleted, substituted or added in the sequence of amino acids shown in Seq. ID No: 4 (“12”).
The present invention also relates to a fusion protein comprising the protein according to any one of “8” to “12” fused with a marker protein and/or a peptide tag (“13”) an antibody specifically bound to the protein according to any one of “8” to “12”( “14”), the antibody according to “14” which is a monoclonal antibody (“15”), a host cell comprising an expression system expressing the protein according to any one of “8” to “12”(“16”).
The present invention also relates to a non-human animal wherein a gene encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is excessively expressed (“17”), a non-human animal wherein a gene function encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is destroyed on a chromosome (“18”), the non-human animal according to “18” having no reactivity against bacterial DNA having an unmethylated CpG sequence (“19”), the non-human animal according to any one of “17” to “19” characterized in that a rodent animal is a mouse (“20”).
The present invention also relates to a method of preparing a cell expressing a protein having reactivity against bacterial DNA having an unmethylated CpG sequence characterized in that the DNA according to any one of “1” to “7” is introduced into a cell wherein a gene function encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is destroyed on a chromosome (“21”), and a cell expressing a receptor protein specifically recognizing bacterial DNA having an unmethylated CpQ sequence obtained by the method of preparing a cell expressing a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence according to “21”(“22”).
The present invention also relates to screening method for an agonist or an antagonist of a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence comprising steps of: in vitro culturing a cell expressing a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence in the presence of a target substance, and measuring/evaluating TLR9 activity (“23”), a screening method for an agonist or an antagonist of a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence comprising steps of administrating a target substance to a non-human animal wherein a gene function encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is destroyed on a chromosome, and measuring/evaluating TLR9 activity of macrophages or spleen cells obtained from the non-human animal (“24”), a screening method for an agonist or an antagonist of a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence comprising steps of: administrating a target substance to a non-human animal wherein a gene encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence is excessively expressed, and measuring/evaluating TLR9 activity of macrophages or spleen cells obtained from the non-human animal (“25”), a screening method for an agonist or an antagonist of a protein having reactivity against bacterial DNA having the unmethylated CpG sequence according to either of “24” or “25” using a mouse as a non-human animal (“26”).
The present invention also relates to an agonist or an antagonist of a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence obtained by the screening method for an agonist or an antagonist of a receptor protein specifically recognizing bacterial DNA having the unmethylated CpG sequence according to any one of “23” to “26”(“27”), a pharmaceutical composition comprising whole or part of a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence as an active component (“28”), a pharmaceutical composition comprising the agonist or antagonist according to “27” as an active component (“29”), a kit used to diagnose diseases related to the deletion, substitution and/or addition in a sequence of DNA encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence comprising the DNA according to “3”, which can compare a sequence of DNA encoding a receptor protein specifically recognizing bacterial DNA having an unmethylated CpG sequence in a test body with a sequence of bases in the DNA according to “3”(“30”).